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Molecular Insight into the Adsorption of Spruce budworm Antifreeze Protein to Ice Surface: A Clathrate Mediated Recognition Mechanism Sandipan Chakraborty, and Biman Jana Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01733 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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Molecular Insight into the Adsorption of Spruce Budworm Antifreeze Protein to Ice Surface: A Clathrate Mediated Recognition Mechanism

Sandipan Chakraborty, Biman Jana*

Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032

*Corresponding author: Dr. Biman Jana: Department of Physical chemistry, IACS, Kolkata-700032, India. Phone: +91 33 2473 4971; Fax: +91 33 2473 2805; E-mail: [email protected]

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ABSTRACT The principal mechanism of ice recognition by antifreeze protein (AFP) is a topic of intense discussion in recent times. Despite of many experimental and theoretical studies, the detailed understanding of the process remains elusive. Present work aims to explore the molecular mechanism of ice recognition by an insect AFP from Spruce budworm, sbwAFP. Evident from our simulation, water dynamics becomes highly sluggish around the IBS due to the combined effect of confinement and ordering induced by the perfectly aligned methyl side-chains of threonine residues, the THRs ladder. The hydroxyl groups of the threonine forms strong hydrogen bonds with few of those highly ordered water molecules that are close to the THRs ladder which is the origin of anchored clathrate water at the IBS of sbwAFP. We propose anchored clathrate mediated basal plane recognition by sbwAFP. The AFP adsorbed on the basal plane through water clathrate framed around the IBS. Surface of the basal plane and anchored clathrate water complete the caging around the threonine residues which is the origin of the binding plane specificity of sbwAFP. This adsorbed AFP-ice complex undergoes dynamic crossover to hydrogen bonded complex within the thermal hysteresis (TH) regime of this particular AFP. The anchored clathrate water becomes the part of the newly grown basal front due to geometrical matches between the basal plane and anchored clathrate water repeat distance. This observation provides structural rationale to the experimentally observed time-dependent increase in TH activity for insect AFP. Our study proposes clathrate mediated ice recognition by AFP and elucidate the dynamic events involve during the ice binding by the insect AFP.

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1. INTRODUCTION Antifreeze proteins (AFPs) help organisms to survive in sub-zero temperature.1 AFP was first identified by Arthur L DeVries in 1969 from the blood of an Antarctic fish2 and later identified from various organisms including several other Antarctic fishes, insects, plants, bacteria and fungi.1,3-15 Cold-adaptation of different organisms in different environments significantly influences the evolution of the protein which resulted in enormous structural diversity among AFPs. They are commonly classified as type I, II, III, IV, insects AFPs and antifreeze glycoproteins.1 Anti-freezing activities greatly vary among different classes of AFPs. Fish AFPs are weakly active and showing thermal hysteresis (TH) ~1 °C16 while insect AFPs show greater degree of TH ~5-6 °C, therefore termed as hyper-active.17 Thermal hysteresis (TH) activity is commonly used to measure antifreezing activity in high AFP concentration range.18 It is defined as non-equilibrium depression of freezing point without altering the melting point. Molecular mechanism of ice growth inhibition is the topic of intense research interest and yet to achieve any consensus view. Initially, direct binding of AFPs to the ice surface mediated by hydrogen bonding was proposed as a possible mechanism of ice recognition. Evident from the crystal structure of several fish and insect AFPs, there are several polar residues, particularly threonine, positioned in such a way that the repeat distances of side-chain hydroxyl groups strongly correlate with the Oxygen repeat distance of that particular ice lattice where it binds.19-21 Initial computer simulation studies in vacuo on a type I AFP also demonstrated that the most preferred binding orientation of the AFP on the [2 0 2 1] pyramidal plane is in [1 1 2] direction where there is matching between the side-chain hydroxyl of threonine residues and ice.21 These observations formed the basis of the hydrogen bond matching hypothesis. However, mutagenesis 3


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studies invalidated the hydrogen bond matching hypothesis. Zhang et al. demonstrated that upon mutation of threonine residues with serine, which is capable of hydrogen bond formation, completely abolish the anti-freezing activity. However, upon mutation with non-polar valine, AFP still remains active.16 Moreover, lately several crystal structures of AFPs have been resolved where the ice binding surface is highly hydrophobic.6,22 Recent simulation studies also demonstrate that AFPs do not interact directly with ice plane rather adsorb on the ice/water interfacial region.

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Therefore, an adsorption-inhibition mechanism has been proposed.

According to this mechanism, AFPs adhere on ice surface and water addition is possible in between two adsorbed AFPs which generates a micro-curvature of the growing ice front. This increases the surface energy which ultimately results in ice growth cessation, known as the “Gibbs-Thomson effect”.1,25,26. The process can be analytically explained using the Langmuir model of impurity adsorption dynamics.27 One of the primary assumptions of this mechanism is that binding of AFP to ice is irreversible. Nature of binding of AFP to the ice surface is another area of intense debate. Studies on the kinetics of AFP adsorption on ice reveal desorption of AFP from ice surface.28,29 However, Pertaya et al., demonstrated that binding of a type III AFP on ice surface is irreversible in nature using fluorescence recovery after photo-bleaching techniques.30 Recent advances in the microfluidic experiments suggest that hyperactive insect AFP remains adsorbed on the ice surface even after removing AFPs from the solution indicating a strong irreversible binding.31 Interestingly, it has been shown that the binding of AFP to ice is a dynamic process and different AFPs exhibit different time dependent TH activity.32,33 Knight and DeVries proposed that competition between the rate of adsorption and ice growth is responsible for the concentration dependence of the TH activity which is very similar to the kinetic pinning theory of Sander and 4


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Tkachenko.34 Hyperactive insect AFP adsorbs on the basal plane in a slow progressive manner.32 Over short exposure time, all hyperactive AFPs showed very low TH activity which increased by a factor of 40 when the exposure time increases to several hour which indicates that there are dynamic phenomenon involved during the AFP adsorption within TH gap.32 However, understanding of the time and concentration dependent TH activity at molecular level is completely lacking. Additionally, AFPs are the topic of immense research interest due to their unusual hydration shell dynamics.35-40 Duboue-Dijon et al. showed that the IBS of a hyperactive insect AFP induces short-ranged water structure enhancement and slowdown of water re-orientational dynamics.41 Low temperature computer simulations of type III and insect AFP reveal formation of highly ordered water around the IBS.42 This preconfigured ordered water has been assumed to help AFP adsorb on ice plane guided by geometrical complementarity.43,44 Ebbinghaus et al., suggested that antifreeze glycoprotein can altered the dynamics of ice/water interface over long distances. Therefore direct binding is not an essential criterion for TH activity.39 However, Celik et al., demonstrated that direct binding is needed to exhibit anti-freezing activity.31 Detailed atomistic insight into the mechanism is clearly lacking which is partly due to the fact that probing the dynamic events involve at the ice/water interface in presence of bound AFP is difficult with experiments and also computationally challenging. Recently, using equilibrium simulation and free-energy calculations we have explored the binding of a type I AFP, wfAFP, to the pyramidal plane.45 We have demonstrated that the AFP adsorbs on the ice plane mediated through the water cage framed around the methyl side-chains of threonine residues. This study for the very first time provides direct rationale to the predicted 5


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role of hydration on the ice binding of AFP. However, the hydration dependent ice binding mechanism is universal to all AFP or it is specific to type I AFP, is yet to be explored. Here, we have investigated the ice binding of an hyperactive insect AFP, Spruce budworm, Choristoneura fumiferana, (sbwAFP), in critical details.9 Particularly, the 501 isoform which is the most active among all other shorter isoforms46 and shows thermal hysteresis up to -6°C. Interestingly, there is a set of highly ordered water strategically located at the IBS. Kuiper et. al., using extensive molecular dynamics has been shown that the ordered water molecules register well with the growing ice front and therefore make sbwAFP bound to the ice plane.47 However, origin of this ordered water is not clearly understood, because ordering can be imposed on crystallographic water due to crystal packing artifacts.43 Also, the detailed molecular understanding of the adsorption of insect AFP in terms of its time-dependent TH activity and irreversible nature of binding as well as the role of ordered water molecules in the process of adsorption are lacking. Here, we have explored the origin of this ordered water molecules present at the IBS. Principal driving forces responsible for the formation of such ordered water on the IBS of sbwAFP has been investigated in critical details. Finally role of these ordered water molecules as well as ice/water interface on the ice recognition has been investigated to provide detailed atomistic insight into the dynamical process involved during the ice binding of AFP. 2. COMPUTATIONAL METHODOLOGY All the molecular dynamics simulations and potential of mean force calculations were performed using OPLS/AA48,49 force field implemented in GROMACS 4.550-52 package. SPC/E53 water model was used to model water and also hexagonal ice slab was built using the same water model. 6


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2.1.

Equilibrium simulation of sbwAFP at different temperatures

3-D structure of the hyperactive antifreeze protein isoform 501 from Spruce budworm (Choristoneura fumiferana) was obtained from Protein Data Bank (PDB ID: 1m8n).46 All heteroatoms including crystallographic water molecules were not considered in the simulation studies. Protein structure was minimized in vacuo using the steepest descent algorithm and then solvated in a cubic box filled with SPC/E explicit water with periodic boundary condition. Box size was so chosen such that all the protein atoms were at least 10 Å apart from the box edges. Three Clions were added to the system to make it charge neutral. Then 500 steps of steepest descent energy minimization followed by 5000 steps of minimization using conjugate gradient algorithm were performed. The minimized solvated protein was then equilibrated using 1 ns position restrained dynamics in the isothermal-isobaric (NPT) ensemble by adding restraining forces on the protein only while the water molecules were allowed to move freely. These position restrained equilibrations were performed in four different temperatures (210 K, 225 K, 250 K and 298 K). Temperature was maintained using an external bath with a coupling constant of 0.1 ps using v-rescale algorithm. The pressure was kept constant (1 bar) by using isotropic Parrinello-Rahman barostat with the time-constant set to 2 ps. Electrostatic interactions were calculated using particle mesh Ewald summation method. Final production simulations of 50 ns were performed in the NPT ensemble at those four different temperatures. During the production run both protein and water molecules are allowed to move freely. The trajectories were stored at every 1 ps. 2.2.

Modeling the binding of sbwAFP to the basal plane

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Molecular docking was used to dock sbwAFP on the basal plane of the ice crystal using a rigid body docking procedure implemented in PatchDock webserver.54 Possible solution, judged by a scoring function, was chosen where the known ice binding site (IBS) of sbwAFP oriented to the ice surface. We performed docking in presence and absence of bound anchored clathrate water to the IBS. Also we performed rigid body docking to obtain a possible docked conformation where sbwAFP docked to the basal plane using its non-IBS. Evident from molecular docking that sbwAFP preferably interacts with the ice plane through plane 3, compared to plane 2. Thus the docking solution where the sbwAFP docked on the basal plane using plane 3 was considered as non-IBS AFP-ice docked complex. 2.3.

Calculation of binding free energy of sbwAFP on basal plane

The docked complexes were then subjected to molecular dynamics simulation. The ice slab in the docked complexes was aligned along the Z-axis. Each system was then solvated in a rectangular water box of 100 Å× 80 Å × 100 Å dimension with periodic boundary condition. The box was so constructed such that in X and Y direction the ice slab did not collide with its image and in Z-direction the box length is greater than double of the final pull distance between AFP and center of mass of the ice slab. Water molecules comprising the ice slab were kept frozen during the binding free energy calculation. Each system was then solvated using SPC/E explicit water and made charge neutral by adding 3 Cl- ions. In case of simulating the sbwAFP-ice complex in presence of bound anchored clathrate water, the anchored clathrate water molecules were also considered explicitly using SPC/E water model. Each system was first minimized with 500 steps using steepest descent algorithm. Then the minimized complex was subjected to 1 ns position restrained dynamics where the ice slab was kept frozen and protein atoms were 8


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restrained, but water molecules were allowed to move freely in NVT ensemble at 225 K using vrescale thermostat with a coupling constant of 0.1 ps. Finally 3 ns NVT simulations were performed for all the AFP-ice docked complexes at 225 K in NVT ensemble before the potential of mean force (PMF) calculation. Umbrella sampling technique was used to calculate binding free energy of sbwAFP on basal ice plane. We considered three different cases: binding of sbwAFP to the basal plane in presence and absence of anchored clathrate water and the other one is the binding of sbwAFP to the basal plane using the non-IBS plane. The center of mass distance along the Z-axis between AFP and ice slab was considered as the reaction co-ordinate and the docked protein was pulled from the ice surface along the Z-direction with an interval of 0.1 nm. An umbrella force constant of 5000 kJ mol-1 nm-2 was used to retain the sbwAFP at desired position. In each umbrella window, 1 ns equilibration followed by 2 ns production run in NVT ensemble was carried out. Weighted histogram analysis method55 available in GROMACS was used to construct the PMF profile. Sufficient overlap among the all the windows was confirmed by histogram analysis. 2.4.

Equilibrium simulation of ice-AFP complexes

The structure of the sbwAFP-ice complex in presence of anchored clathrate water which corresponds to the PMF minimum was then subjected to 30 ns equilibrium simulations at 225 K in NVT ensemble using the same parameter mentioned above. In the entire equilibrium simulations ice slab was kept frozen in all the three dimensions. A representative structure of the sbwAFP-ice complex in presence of anchored clathrate water obtained after 10 ns equilibrium simulation mentioned above was then subjected to another

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equilibrium simulation at low temperature (210 K) in NVT ensemble using the same simulation parameters mentioned above.

3. RESULT AND DISCUSSION Spruce budworm, Choristoneura fumiferana, (sbwAFP) 501 isoform shows highest degree of thermal hysteresis among all the AFP isoforms which has been ascribed to the presence of higher number of β-helical coil in its structure, a phenomenon commonly termed as loopectomy.46 The protein is a β-helix with a triangular cross-section and each side is composed of parallel β-sheets. Structure of the protein is shown in Figure 1. The ice binding plane is referred as plane 1 whereas plane 2 and 3 are the non-ice binding planes (Figure 1A). The ice binding surface contains array of threonine residues, here referred as THRs ladder. All the threonine residues are located on the IBS, except THR 12. THR 12 is located at the cross-section of plane 2 and 3 and referred as non-ice binding THR throughout the text (Figure 1B). In the IBS, there are six crystallographically resolved water molecules that are found to form hydrogen bonds with the THRs ladder (Figure 1C).

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Figure 1: Structure of Spruce budworm, Choristoneura fumiferana, (sbwAFP) 501 isoform. (A) Cartoon representation of the AFP where ice binding plane and different other non-ice binding planes are shown. (B) Position of threonine residues are shown in green stick mode. The array of threonine residues at the ice binding surface is referred as THRs ladder and the non-ice binding threonine residue (THR 12) is also shown. (C) Detail insight of the ice binding surface of the sbwAFP. All the threonine residues are shown in green sticks and the ordered waters are shown in orange sphere representation.

Due to an excellent geometrical match between these ordered water and the ice plane where it binds, the ordered water molecules has been implicated to play pivotal role in anchoring the AFP to the ice plane. This mode of binding is commonly referred as “anchored clathrate” mechanism.43 Here, we have analyzed the molecular origin of the ordered water molecule and its 11


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functional relevance in terms of ice binding ability using equilibrium simulation and free energy calculations. 3.1.

Dynamics of water around the ice binding surface of sbwAFP: Origin of anchored clathrate water

We first characterize the nature of IBS of sbwAFP and compare with the non-IBS planes using electrostatic surface potential calculations with the aid of the Poisson-Boltzmann equation solver implemented in the CHARMM-GUI web-interface56,57 and results are shown in Figure 2.

Figure 2: Electrostatic surface potential of different planes of sbwAFP. Blue and red colors are used to indicate the most positive and negative electrostatic potentials, respectively.

The IBS of the sbwAFP is flat and less polar compared to the non-IBS surfaces. Notably, the plane 2 and 3 contain many serine residues along with other charged amino acid residues. Also, 12


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both the non-IBS planes are not flat rather possess several crevices. Strategically located THRs ladder is consecutively oriented in such a way that it provides flatness to the IBS which can register well to the nearly flat basal ice plane. Apart from providing flatness to the IBS, role of the THRs ladder on the ordering of hydration water around the IBS and its temperature dependence have been explored further using equilibrium simulations. We have considered only single AFP without any crystallographic waters in the simulation box. Hydration dynamics around the IBS has been studied using 50 ns equilibrium simulation at four different temperatures (210 K, 225 K, 250 K and 298 K). It is noteworthy that the melting temperature (Tm) of SPC/E water is 215 K. Hydration of the IBS of sbwAFP has been inferred in terms of the threonine residues and compared with the non-IBS threonine, serine residues of plane 2 and 3, respectively. Analysis of the radial distribution function of water molecule (Oxygen) around the hydroxyl group of threonine (IBS and non-IBS) or serine (plane 2 and 3) reveals that there is preferential distribution of water around the threonine hydroxyl groups at the IBS in all four different temperatures (Figure 3AI-DI). Oxygen (threonine/serine)-Oxygen (water) distance is 0.27 nm indicating that the water is strongly hydrogen bonded with IBS threonine residues. Irrespective of temperature, water distribution around IBS threonine is almost double compared to non-IBS threonine or serine residues of plane 2 and 3. We have further characterized the AFPwater interaction using the hydrogen bond autocorrelation function at four different temperatures (Figure 3 AII-DII).

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Figure 3: Radial distribution function of water (Oxygen) molecules around the side-chain hydroxyl (Oxygen) of sbwAFP threonine (IBS and non-IBS) or serine (plane 2 and 3) at 210 K (AI), 225 K (BI), 250 K (CI) and 298 K (DI) is shown. Hydrogen bond autocorrelation function analysis of water and side-chain hydroxyl (Oxygen) of sbwAFP threonine (IBS) or serine (nonIBS) at 210 K (AII), 225 K (BII), 250 K (CII) and 298 K (DII) is shown.

The dynamics of the hydrogen bond between water and the THRs ladder of the IBS is markedly slow compared to the non-IBS planes. Hydrogen bond autocorrelation function has been successfully deconvoluted by using a bi-exponential decay function. 14


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Table 1: Hydrogen bond life-times of AFP-water at different planes and at different temperatures calculated with the Luzar-Chandler analysis have been listed. Temperature 210K

225K

250K

298K

AFP planes

Hydrogen bond lifetime (ps)

IBS

16.76

Plane 2

10.39

Plane 3

8.30

IBS

12.73

Plane 2

6.89

Plane 3

6.74

IBS

8.93

Plane 2

5.98

Plane 3

4.91

IBS

4.87

Plane 2

3.6

Plane 3

3.1

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At 210 K, below the Tm of SPC/E water, water dynamics in the hydration layer is highly slow which gives rise to a slowly decaying tail. This nature of decay is extremely slow around the IBS compared to the other two non-IBS planes of sbwAFP. With increasing temperature, the decay time of the slowly decaying component decreases. However, around the IBS, the dynamics is almost twice as slow as compared to the non-IBS planes. Further hydrogen bond lifetimes have been calculated using the theory of Luzar and Chandler and the calculated average lifetimes of the hydrogen bonding interactions between AFP and hydration water have been summarized in Table 1. The hydrogen bond life-time of AFP-water at the IBS plane is almost ~ 1.5-2 times higher compared to the non-IBS plane in all the four different temperatures. This feature is interesting because our electrostatic surface potential calculation reveals that non-IBS planes of sbwAFP are more polar comparative to the IBS plane. However, IBS forms stronger hydrogen bonds with water compared to the non-IBS planes. This can be ascribed by two factors. Firstly, the IBS is mostly composed of threonine residues while the non-IBS hydrogen bonding residues are serine. Threonine is known to form stronger hydrogen bonds with water compared to serine due to steric confinement effect. The second one which is probably the most important one is the cooperativity induced by the presence of series of threonine residues that are perfectly aligned at the IBS. We have then analyzed the effect of methyl groups of THRs ladder on the water structure formation around the IBS using radial distribution function. For comparison we have also considered the distribution around the non-IBS threonine and results are shown in Figure 4 AICI. At 298 K, there is no such preferential water density around the methyl side-chain (data not shown) in the first solvation layer. However, in all other temperatures there is preferential water density around the methyl group of threonine within the first hydration shell. Particularly at 210 16


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K and 225 K, water density is higher around the methyl groups of the THRs ladder compared to the non-IBS threonine. However at 250 K, they become indistinguishable with a little preference over the non-IBS threonine. It is pertinent to mention that the melting temperature of SPC/E water is 215 K. Therefore, 210 K and 225 K can be considered as working temperature of the AFPs. In this temperature region the hydration of hydrophobic methyl side-chains of threonine becomes significant around the IBS of sbwAFP which induces preferential water distribution.

Figure 4: Radial distribution functions of water (Oxygen) molecules around the side-chain CH3 (Carbon) of sbwAFP threonine (IBS and non-IBS) at 210 K (AI), 225 K (BI) and 250 K (CI) are shown. O-O-O angle distributions among water molecules within 5.5 Å of the threonine CH3

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groups of IBS at three different temperatures, 210 K (AII), 225 K (BII) and 250 K (CII) are shown.

We have further analyzed the water structure formation around the methyl group of THRs ladder of the IBS of sbwAFP. O-O-O angle distribution of water in the first layer of solvation shell around the methyl groups of THRs ladder i.e., ~ 0.55 nm (evident from radial distribution plot), has been used to probe water ordering. This angle distribution is a convenient order parameter describing tetrahedral geometries of water hydrogen bond network.58,59 The distribution is characterized by two peaks; one centered ~ 100-108° describing tetrahedral water whereas second one, ~ 60°, is used to probe interstitial water. Notably in the low temperature region, water molecules within the first solvation shell around IBS is preferentially distributed in tetrahedrally coordinated hydrogen bonded network, compared to bulk. Evident from the Figure 4 AII-CII, the distribution is shifted from ~ 100° in case of bulk to ~ 107-108° around the first solvation shell around the IBS. Thus ~ the Tm of SPC/E water model, there is preferential distribution of highly ordered water network around the hydrophobic IBS of sbwAFP. We have further characterized the hydrogen bonding interactions of these order water network around the IBS and results are shown in Figure 5.

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Figure 5: (A) Hydrogen bond autocorrelation function of water within 5.5 Å of the side-chain CH3 (Carbon) of sbwAFP threonine (IBS) or Cβ (Carbon) of non-IBS serine at 210 K, 225 K, and 250 K. H-O-O angle distribution among water molecules within 5.5 Å of sbwAFP threonine (IBS) or serine (non-IBS) at 210 K (B), 225 K (C), and 250 K (D). (E) Water organization around the IBS of sbwAFP is shown. Protein is rendered in cartoon and the THRs ladder is shown in stick representation. Water around the IBS is shown in line mode and hydrogen bonding is shown as red dotted line. Water molecules occupy the anchored clathrate water position are shown in CPK representation.

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The dynamical aspect of this co-ordinated hydrogen bonded network has been probed further using the hydrogen bond autocorrelation function and results are depicted in Figure 5A. From this autocorrelation function, hydrogen bond lifetimes have been calculated using the Luzar and Chandler analysis and summarized in Table 2.

Table 2: Hydrogen bond life-times of the hydrogen bonded network of water molecules within 5.5 Å of the side-chain CH3 (Carbon) of sbwAFP threonine (IBS) or serine (non-IBS) at 210 K, 225 K, and 250 K calculated with the Luzar-Chandler analysis have been listed. Temperature

AFP planes

Hydrogen bond lifetime (ps)

210K

IBS

9.31

Plane 2

7.78

Plane 3

7.34

IBS

7.07

Plane 2

6.11

Plane 3

5.79

IBS

5.03

Plane 2

4.59

Plane 3

4.10

225K

250K

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Evident from the table, hydrogen bond lifetime of the water network in the first solvation shell around the IBS is higher compared to non-IBS plane which implies strong hydrogen bond formation between water solvating the IBS of sbwAFP. At 210 K and 225 K, this distinction is pronounced, however, at 250 K the difference between the hydrogen bond lifetimes of the water network around the IBS becomes less significant compared to non-IBS (plane 2). The hydrogen bond lifetime data has been further complemented with the Hydrogen-Donor-Acceptor (H-O-O) angle distribution calculations at three different temperatures of first hydration shell water molecules around both IBS and non-IBS. H-O-O angle distribution is a useful order parameter and was previously used to probe water structure ordering around the AFP. The distribution has a characteristic bi-modal distribution when 4 Å Donor-Acceptor distance cut-off has been consider.60 The low angle peak ~ 7° implies strong (linear) hydrogen bond while the large angle peak represents weak hydrogen (bend) bonds. Interestingly, at 210 K and 225 K, population of ordered water molecules connected through strong hydrogen bonding interactions within the first hydration shell of IBS is higher compared to the non-IBS. At 250 K, the population of strong hydrogen bonding water network in the first hydration shell is almost comparable with the distribution observed in case of plane 2, however, it is higher compared to plane 3. Coherence between all the data reinforces the fact that at 210 K and 225 K, hydration of hydrophobic methyl side-chains of THRs ladder present at the IBS of sbwAFP becomes highly significant. Considering the fact that the Tm of SPC/E water is ~ 215 K, therefore the 210-225 K is the temperature region where AFPs are expected to be active. Strategically oriented methyl side-chains of the perfectly aligned threonine residues at the IBS enhance the ordering of the hydration water around the methyl side-chains within the first hydration shell. Such perfectly aligned THRs ladder induces flatness at the IBS and therefore water dynamics becomes slower 21


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due to confinement. Due to the combined effect of ordering and slow dynamics, the hydroxyl groups of the THRs ladder form strong hydrogen bonds with water. Structure of first hydration shell solvating the IBS of sbwAFP is shown in Figure 5E. Interestingly, although we have started the simulation without the crystallographic water however during the simulation, six water molecules occupy similar positions of the anchored clathrate water in the IBS as observed in the crystal structure of the sbwAFP (Figure 1C). Most of these waters are doubly hydrogen bonded with two nearby threonine residues and also is a part of a highly ordered tetrahedral hydration water network. Therefore, the presence of the anchored clathrate water at the IBS is primarily contributed by the flatness and hydrophobicity of the IBS surface. 3.2.

Role of anchored clathrate water on ice adsorption by sbwAFP: Potential of mean force calculation

We now explicitly determine the role of this anchored clathrate water on the adsorption of sbwAFP on ice surface using the potential of mean force (PMF) calculation. Our objective is to estimate the binding free energy of AFP to the ice surface at the point of initial contact. During this initial recognition process, we need to consider the binding of AFP to the ice surface through an ice/water interface. Previously Bryk et al., demonstrated that the basal and prism plane of ice interfaces can be appropriately mimicked by SPC/E water at the temperature 225 ± 5 K.61 Thus we have performed the PMF calculation at 225 K and we have considered the basal plane of ice. It is known the sbwAFP strongly adsorbs on the highly growing basal plane which can be accounted for the observed high anti-freezing activity of the protein. We have calculated the potential of mean force of sbwAFP on basal plane with and without anchored clathrate water and compared it with the non-IBS adsorption of sbwAFP on the basal plane. Initial docked complex 22


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of AFP on the basal plane has been obtained from a rigid body docking procedure implemented in the PatchDock Server.54 Interestingly, the lowest energy docked complex is the one where the IBS of sbwAFP directly hydrogen bonded to the basal plane of the ice slab. Structure of this initial docked complex is shown in Figure S1 (Supporting information). For comparison, we have considered the binding of the non-IBS plane of sbwAFP to the basal plane. Binding free energies of both IBS and non-IBS of the sbwAFP on basal plane have been calculated using umbrella sampling method and the results are shown in Figure 6.

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Figure 6: PMF profile for the binding of sbwAFP to the basal plane of ice through the ice binding surface in presence of anchored clathrate water (black), in absence of anchored clathrate water (green) and also binding of sbwAFP to the basal plane through the non-IBS (yellow). Error estimation was obtained from the bootstrap analysis. Closer insight into the minima of the PMF profile of the binding of non-IBS sbwAFP to the ice plane is shown in the inset.

Interestingly, although we have started from a state where the THRs ladder of the IBS is hydrogen bonded to the ice surface, characterized by a center of mass (COM) distance between AFP and ice (along the Z-axis) is ~ 1.55 nm, however the minimum in the PMF profile corresponds to a bound state where the COM distance between the AFP and ice is ~ 1.64 nm. Interestingly, when the anchored clathrate was not considered in the PMF study, the binding affinity reduces (green line) compared to the adsorption of AFP on ice surface in presence of bound anchored clathrate water (black). However there is no change in the position of the minima in the PMF profile. In case of the non-IBS of sbwAFP (plane 3) binding to the basal plane, there are two minima in the PMF profile. The minima-I has been observed at 1.52 nm COM distance from AFP to ice while the minima-II has been found to be at 1.56 nm (AFP and ice COM distance along the Z-axis). Structures of both the minima are shown in Figure S2 (supporting information). Evident from the figure that the bound states of the non-IBS of the sbwAFP to the basal plane are hydrogen bonded complex. Many polar residues, i.e., GLN 16, LYS 19, ASN 47, LYS 49, TYR 64 and TYR 77 are primarily involved in the hydrogen bonding interactions in both the minima. Clearly evident from the PMF plot that the most favorable bound state of sbwAFP is a state where the AFP is adsorbed at the ice/water interface through the 24


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IBS in presence of anchored clathrate water. Structure of the adsorbed sbwAFP-ice is shown in Figure 7. To further reinforce our observation we have performed the free energy calculation using another water model, TIP4P and a comparison of the PMF profiles of AFP adsorption on the basal plane through the IBS in presence of anchored clathrate water is shown in Figure S3 (supporting information). As evident from the PMF profile, the position of the minima remains unaltered in both water models which further strengthen our observation that the AFP is not forming direct hydrogen bonds with ice surface rather adsorbed at the ice/water interface. However, the affinity of adsorption reduces by changing the water model from SPC/E to TIP4P. It is due to the effect of temperature at which the simulation has been performed. The Tm for SPC/E and TIP4P are 215 K and 232 K, respectively. Therefore, we performed free energy simulations at 225 K and 240 K using SPC/E and TIP4P water model, respectively. Closure insight into the structure of the sbwAFP-ice complex at the PMF minima reveals that most of the hydrogen bonding interactions between THRs ladder and ice surface are lost rather the protein is adsorbed on the ice plane. Interestingly, the methyl side-chains of THRs ladder orient towards the ice plane and there is a significant ordering of ice-water interfacial water around the methyl groups in this bound state. This binding mode implicates that there is a significant role of hydrophobic hydration on the adsorption of sbwAFP on ice surface. Specific orientation of perfectly aligned methyl side-chains of THRs ladder at the IBS significantly induces cage like water structure formation around the IBS that help AFP adhere on the ice surface. Notably, the ice surface frames edges of the water cages around the IBS. Surface of the basal ice plane is so perfectly aligned that it can complement the formed water cages which enlighten the observed binding specificities of sbwAFP towards the basal plane.

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Figure 7: Structures of the sbwAFP-ice complex corresponds to the PMF minima. (A) Front view of the bound state of the complex is shown where the protein is shown in cartoon representations and THRs ladder is shown as cyan sticks. Ice plane is represented in vdW representation. (B) A closer look into the binding details of the THRs ladder at the IBS of sbwAFP to the basal plane through the anchored clathrate water is shown. Anchored clathrate water is shown in CPK mode and ice surface and ice/water interfacial water are shown in line mode. Hydrogen bonds are shown as red dotted lines. (C) Side view of the bound state of the sbwAFP-ice complex is shown where the protein is shown in cartoon representations and THRs ladder is modeled as cyan vdW mode. Ice and ice/water interfacial water surrounding the THR

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ladder is shown in stick representation and hydrogen bonding interactions are shown as red dotted lines.

Apart from this hydrophobic hydration driven binding, hydrogen bonding also plays crucial role on the adsorption of AFP to the ice surface. The hydroxyl side-chains of the aligned threonine residues of the THRs ladder orient inwards in the bound conformation. There they find the anchored clathrate water which forms efficient hydrogen bonds with both the hydroxyl groups of THR ladder and ice surface. These strategically oriented anchored clathrate water molecules form a cooperative hydrogen bonding network between AFP and ice plane which makes the caging around the THRs ladder complete. In this adsorbed form the anchored clathrate water molecules are engaged in highly ordered water network that anchored AFP to the ice surface. This binding is further complemented from the PMF profile of the binding of sbwAFP to the ice surface in absence of anchored clathrate water. Evident from the figure, in absence of the anchored clathrate water, binding affinity of the sbwAFP towards the basal plane reduces significantly. Closer insight into the structure corresponding to the PMF minima (Figure S4, supporting information) reveals that the binding orientation is very similar to the sbwAFP adsorption on the basal plane in presence of anchored clathrate water. Similar THRs ladder driven water cage mediated AFP adsorption on basal plane has been observed, rather than hydrogen bonding complex. However, due to the absence of the anchored clathrate water, the caging around the THRs ladder becomes incomplete which significantly reduces the binding affinity. During the simulation, only a single water molecule occupies the anchored clathrate water position in the sbwAFP-ice complex (Figure 8). 27


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Figure 8: Close view of the water organization around the IBS of the sbwAFP-ice complex structure corresponds to the minima in the PMF profile of the ice binding of sbwAFP to the basal plane through the ice binding surface in absence of anchored clathrate water. Protein is rendered in cartoon and the THRs ladder is shown in cyan stick representation. Water around the IBS is shown in stick mode and hydrogen bonding is shown as red dotted line. The single water molecule that occupies the anchored clathrate water position during the simulation is shown in CPK representation.

We have further performed 30 ns equilibrium simulations at 225 K of the adsorbed state of the AFP-ice complex in presence of bound anchored clathrate water to infer the stability of the state and also further complement the ice binding free energy results. Throughout the simulation timescale AFP remains adsorbed on the ice plane which signifies high stability of the adsorbed state. During the equilibrium simulation the anchored clathrate water is highly stable with residence time ~ 20 ns, indicating high stability of the co-ordinated hydrogen bonded network 28


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that anchored AFP to the basal ice plane. After 20 ns, an anchored clathrate water moves a little bit but the overall anchored clathrate geometry remains stable throughout the simulation.

Figure 9: (A) Mean square displacement (MSD) of anchored clathrate water in the sbwAFP-ice adsorbed complex during simulation time scale, rest of the water has been considered for reference. (B) Radial distribution function (RDF) of water (Oxygen) molecules around the sidechain CH3 (Cγ) of THRs ladder in sbwAFP-ice complex during simulation time. RDF plots of 29


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ice/water interfacial water (blue), ice/water interfacial water with anchored clathrate water (red) and ice/water interfacial water with anchored clathrate water and ice slab (black) are shown. (C) H-O-O angle distribution among water molecules (Ice water molecules were excluded) within 5.5 Å of the CH3 groups of threonine at the IBS (black) and non-IBS (red) of sbwAFP-ice complex during the simulation timescale and compared with the bulk (blue) distribution. (D) Donor-acceptor distance distribution among water molecules (Ice water molecules were excluded) within 5.5 Å of the CH3 groups of threonine at the IBS (black) and non-IBS (red) of sbwAFP-ice complex during the simulation timescale and compared with the bulk (blue) distribution. (E) Hydrogen bond autocorrelation function of THRs ladder of IBS-anchored clathrate water (black) and ice-anchored clathrate water (red) calculated from the equilibrium simulation are shown and compared with the non-IBS threonine-water (blue).

Diffusion of the anchored clathrate water is highly slow with respect to other water molecules at that particular temperature (Figure 9A). Theses water molecules are anchored with AFP on the basal ice plane through highly co-operative hydrogen bonding network as well as they are trapped within AFP and ice plane, therefore they are highly confined. These confinements provide motional restriction and therefore the cooperative hydrogen bonding network between AFP and ice mediated through these anchored water becomes highly stable. Analysis of radial distribution function around the Cγ of the THRs ladder of the IBS in the adsorbed state reveals an interesting feature (Figure 9B). In low temperature due to hydrophobic hydration there is formation of water structure evident from preferential distribution of water within the first solvation shell. The peak appears around 0.37 nm from the Cγ of the methyl side-chains of the 30


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THRs ladder in the bound state (Blue line). Interestingly, when we have incorporated anchored clathrate water in our analysis only the peak ~ 0.37 nm increases significantly (red line). Further consideration of the ice plane reveals further increase of the peak ~ 0.37 nm (Black line). These features indicate that both anchored clathrate water and ice surface are taking part to complete the framing of the water cage around the methyl side-chains of the THRs ladder of the IBS formed by the ice/water interfacial water in the first solvation shell. The appearances of defined structures in higher distances from the Cγ of the THRs ladder in the black line in the radial distribution function is due to the presence of ice slab where the water molecules are retained in a highly ordered orientation. Nature of the hydrogen bonds in these water cages around the IBS of sbwAFP has been probed by the H-O-O angle distribution (Figure 9C). Interestingly, within the first solvation shell around the methyl group, water molecules are involved in strong hydrogen bonding network compared to the bulk. H-O-O angle distribution is almost confined in the low angle region with the characteristic donor-acceptor distances of ~ 0.28 nm (Figure 9D), indicating involvement of very strong hydrogen bonding network of the water cage around the IBS in the adsorbed state. Interstitial water molecules that might affect the cage like structure of the water network are significantly less around the methyl group compared to the bulk. Dynamical aspects of this water network have been probed further with hydrogen bond autocorrelation function and the results are shown in Figure 9E and summarized in Table 3. Hydrogen bond autocorrelation function has been successfully deconvoluted into tri-exponential decay. Timescales of the three components greatly varies: one is in the sub-picosecond range, another is in the range of 10-100 ps and the third one is in the nanosecond range. Interestingly, 31


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the timescales of different components and their fractional contribution is almost similar in case of THRs ladder-Anchored clathrate water hydrogen bonds and ice-anchored clathrate water hydrogen bonds which imply that these two hydrogen bonds are highly cooperative in nature.

Table 3: Dynamical Properties of the hydrogen bonded network of the adsorbed AFP-ice complex is tabulated. Hydrogen bond orientational correlation times (τ1, τ2 and τ3) has been listed. Systems

Hydrogen bond autocorrelation time scale (ps) a1

τ1

a2

τ2

a3

τ3

IBS THRs-Anchored clathrate water

0.44

0.37

0.05

94.34

0.51

5028.83

ICE-Anchored clathrate water

0.49

0.56

0.15

27.42

0.36

2235.67

Non-IBS THRs-water

0.15

0.56

0.13

75.68

0.72

947.24

However, the dynamics of the hydrogen bond between the non-IBS THR and water is markedly different, particularly the time scale of the slow component. Thus the equilibrium simulation further reinforces the fact that anchored clathrate water molecules is a part of the water cages that encapsulated the THRs ladder at the IBS and help AFP to efficiently anchor to the basal plane.

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3.3.

Dynamical transition of the adsorbed ice-AFP complex to a hydrogen bonded complex and its functional relevance

Till now we have considered the initial recognition process where AFP first interacts with the ice plane through the ice/water interface. However, to understand the antifreezing activity of sbwAFP, we need to understand the effect of the adsorbed AFP on a growing ice front within the TH regime. Drori et al., showed that TH dynamics of hyperactive insect AFP is markedly different than that of type III AFP.32 Insect AFP shows gradual increase in TH activity with time.32 To probe this dynamical phenomenon we have simulated the adsorbed sbwAFP-ice at 210 K, considering the fact the Tm of SPC/E water is 215 K. It is noteworthy that this is the working temperature of the sbwAFP, since the AFP shows thermal hysteresis of 5-6 °C. We have monitored the structure of the water cages around the THRs ladder of the IBS in the bound state using F4 order parameter (Figure 10). For comparison we have considered the dynamics of the first solvation water around the non-IBS threonine at both 225 K and 210 K. F4 is a highly sensible water parameter that has been devised to distinguish between clathrate and ice.62-64 The average values of F4 order for ice, clathrate and liquid water are -0.3, 0.7 and -0.04, respectively.62

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Figure 10: Analysis of F4 order parameter distribution of water around the THRs ladder of the IBS of sbwAFP-ice adsorbed complex from equilibrium simulation at 225 K (black) and 210 K (blue). For comparison distribution of the F4 order parameter of water around the non-IBS threonine also has been considered at 225 K (yellow) and 210 K (red).

Typically the water structure around the non-IBS threonine can’t be characterized as either clathrate like or ice-like, rather a broad distribution of the F4 order has been observed. This feature is in line with the Cheng et al., who have demonstrated that surface topography significantly influences the hydration structure around the hydrophobe and ideal clathrate orientation is not possible rather the water structure shows fluctuating distribution of clathratelike and other orientationally inverted structures.65 Interestingly, the distribution pattern remains broad-like upon lowering the temperature from 225 K to 210 K, with a marginal increment of 34


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ice-like water. However, the distribution of F4 order parameter markedly changes in case of THRs ladder at the IBS when adsorbed on ice surface. Structure of the water cages surrounding the THRs ladder of the IBS in the adsorbed state is mostly clathrate-like, evident from the 10 ns equilibrium simulation of the adsorbed state at 225 K. We did not consider the ice slab in the F4 order parameter calculation; since the ice slab is already preconfigured in the ice-1h form therefore can induce bias in the F4 value. Presence of water clathrate around the THRs ladder in the adsorbed state can be rationalized in terms of both the effect of a flat hydrophobic IBS where threonine residues are perfectly aligned which can induce cooperativity and also the confinement induced by the nearby ice plane. Thus water molecules around the THRs ladder are more ordered and also motionally restrained which might induce clathrate formation. However, upon lowering the temperature at 210 K, the working temperature of the sbwAFP, there are marked changes in the F4 order distribution. The water cages around the THRs ladder are mostly ice-like and clathrate like water molecules in the first solvation shell around the IBS reduced significantly. Thus there is a transition of the structure of the water cages surrounding the IBS THRs ladder from clathrate-like to ice-like water upon going below the Tm. This behavior has functional significance and in line of the Gibbs-Thomson effect. In the working temperature of the AFP, i.e., below the Tm, the ice/water interface is almost ice-like which induces structural transformation of the water clathrate structure into ice-like ordered water layer. Thus, the adsorbed AFP-ice complex undergoes a dynamic crossover from a water clathrate mediated adsorbed complex to a hydrogen bonded complex during the growth of the ice front. Anchored clathrate water repeat distances closely match with the oxygen repeat distance of the basal plane surface. Upon further growth of the ice front of the basal surface, the anchored clathrate water becomes part of the growing ice front. Therefore, the bound AFP forms direct hydrogen bonds to 35


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the ice front of the basal plane. Now, water addition is only possible surrounding the bound AFP leading to the curvature of the growing ice front. Our simulation observations and predictions are consistent with the Gibbs-Thompson effect.24,47 and in accordance with the recent simulation studies which showed that the binding of sbwAFP on ice surface affect local ice growth kinetics.66 The observed dynamic transition from the clathrate-mediated bound state to hydrogen bonded complex within the hysteresis regime is the origin of the experimentally observed timedependent TH activity. Notably, Wilson et al., monitored the ice/water interface during the accumulation of anti-freeze glycoprotein (AFGP) on ice surface using ellipsometry techniques and observed that the interface changes over time and reached a plateau after 60 min which strongly complement our observation.67 Here, we have tried to interpret the phenomenon by mimicking the process. Initially, we have probed AFP binding to an ice crystal in presence of ice/water interface by performing a PMF calculation at 225 K. Notably, using the SPC/E water model at 225 K there is no ice growth observed during the simulation timescale. Our simulation reveals water clathrate mediated AFP adsorption on basal ice plane. Then we have probed the water structure transformation around the IBS of the AFP in the adsorbed state when there is growth of ice layer and the process has been mimicked by lowering the simulation temperature at 210 K. There we find a structural transition in the TH regime where the adsorbed complex transformed into the hydrogen bonded complex. We have mimicked the conditions and interpret the structural transition in terms of ensemble average F4 order parameter. However, probing the dynamical transition of the adsorbed complex to hydrogen bonded complex is a long time phenomenon occurs in the timescale of hours67 which is intractable computationally. Thus conclusions regarding the rate of this dynamical process are beyond the scope of this study.

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4. CONCLUSIONS Despite immense efforts using different experimental techniques as well as theoretical approaches, the molecular origin of ice recognition by antifreeze protein remains highly controversial. Several competing hypothesis have been proposed but none of them is conclusive enough to explain experimental observations. Present study aims to explore a unified molecular mechanism of ice recognition by AFP. Here, we have investigated detailed dynamic process involved in the ice recognition by a hyperactive insect AFP from Spruce budworm, sbwAFP isoform 501 which shows thermal hysteresis up to -6 °C46 using equilibrium simulation and binding free energy calculations. We have probed the origin of anchored clathrate water and our simulation shows that the anchored clathrate water present at the IBS of insect AFP is solely due to the orientation of the THRs ladder and hydrophobicity of the IBS, not due to crystal packing artifacts. At low temperature, hydration of hydrophobic methyl side-chains of perfectly aligned threonine residues at the IBS enhances ordering of the hydration water. Such perfectly aligned THRs ladder also induces flatness to the IBS which further retards water dynamics due to confinement. Among these slowly diffusive water molecules, those water molecules that are capable of forming hydrogen bonds with the hydroxyl groups of the THRs ladder becomes the anchored clathrate water. Recently, synergism of hydrophobic and hydrophilic interactions also has been assumed to play major role in formation of ice-like ordered water shell around the IBS of Tenebrio molitor AFP68 and also around the surface of ice-nucleating bacterial protein from P. syringae.69 These anchored clathrate water molecules plays major role in basal plane recognition by sbwAFP. Potential of mean force calculation reveals that the minimum in the binding free energy 37


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profile corresponds to a state where the AFP is adsorbed on the basal plane through a network of highly ordered water around the THRs ladder of the IBS. Perfectly aligned methyl side-chains of THRs ladder induce water clathrate formation around the THRs ladder. Surface of the basal plane and strategically oriented anchored clathrate water also becomes part of this water clathrate around the IBS and makes the caging around the IBS complete. Anchored clathrate water makes co-operative dual hydrogen bonds with both AFP and ice which efficiently adsorb sbwAFP on the ice plane. Synergy among the oxygen repeat distances of the basal ice surface, position of the anchored clathrate water molecules and water cage structures around the methyl groups of the THRs ladder makes the structure of the water clathrate complete around the IBS. This binding mode is in accordance with the adsorption mechanism of type I AFP on pyramidal plane45 which implicates the possibilities of a unified mechanism of ice recognition by AFPs. Further investigations of other groups of AFPs are needed to conclude on this aspect. Also this water cage mediated AFP adsorption mechanism links between the experimentally observed unusual hydration of AFP to the absorption inhibition model. Interestingly, we have observed that within the TH regime, there is a dynamic crossover of the ice/water interfacial water sandwiched between adsorbed AFP and ice surface from clathrate to ice like. This behavior has functional significance and supports irreversible ice binding mechanism.30-33 In the working temperature of the AFP, i.e., below the Tm, the ice/water interface is almost ice-like which induces structural transformation of the water clathrate around the IBS of adsorbed AFP to ice-like ordered water layer. Therefore with further growth of a single layer on top of the existing ice surface, the anchored clathrate water becomes part of the growing ice surface thus the adsorbed AFP now forms direct hydrogen bonding with the newly grown ice surface and becomes strongly bound. Our study for the first time provides the atomistic insight into the time-dependent TH activity 38


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observed in case of insect AFP. The observation is very well complemented by Wilson et al., who demonstrated that ice/water interface changes over time during the accumulation of AFP on the ice surface.67 Also the irreversible direct binding of insect AFP on basal plane justifies the observation that hyperactive AFPs remains bound to the ice surface even removing the surrounding AFPs from solution which implicates a strong irreversible binding.31

Supporting Information Additional supplementary figures are provided as supporting information (ESI). This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This research is funded by the Department of Science and Technology SERB grant EMR/2016/001333. Authors also gratefully acknowledge the central supercomputing facility (CRAY) at Indian Association for the Cultivation of Science, Kolkata. SC acknowledges all the lab mates for stimulating discussions, particularly Mr. Sridip Parui for his help during the development of the order parameter code.

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